Week 2: DNA REPLICATION - DNA Replication Machinery Flashcards
What does semi-conservative mean ?
Each molecule has an original and a new strand
What synthesises DNA?
DNA polymerase
What phase of the cell cycle does DNA rep occur?
S-PHASE
8hrs
What does DNAp require to synthesis new DNA strand?
all the (A,T,G,C) deoxyribonucleoside triphosphates,
Mg2+,
3’ OH paired to DNA template
What direction is continuous DNA rep?
5->3
What side are DNA bases continually added?
3’
What is the byproduct of DNA polymerisation?
di-phosphate group
What is the site where DNA rep starts?
origin
How is the 3’->5’ direction replicated?
in fragments, discontinuous.
called Okazaki fragments
Draw the structure of the DNA backbone…
ANSWER
What is chromatin?
Highly conserved DNA
Many nucleosomes that create a chromatin fibre and eventually a chromosome
What makes a histone?
octamer
4 x dimers
How much DNA can an octamer of HIS wrap?
~1.7 turns or ~147 bases
What are the HIS presenting a DNA octamer?
H2A, H2B, H3, H4
What makes nucleosome?
An octamer of Positive HIS proteins (core)
negative backbone of DNA wraps around positive His.
“bead”
What alters the structure and number of nucleosomes?
Chromatin remodelling Enzymes
What can recruit chromatin remodelling enzymes?
Transcription activators
bind to enhancer sequences
What is an issue with nucleosomes and DNA rep?
hinderance at the origin.
machinery can’t access it
What is dormant origin, when are the activated?
activated when 2 chain are about to meet and an origin lies in the middle.
It allows no bases to be missed and chains can meet
ligase enzyme binds the 2 chains together
How many replication origins does EUK vs PRO have?
PRO = 1 EUK = clusters (many)
What 3 essential elements are needed at the replication origin?
- BS for initiator/ O rep P
- Start site for rep
- Elements for unwinding/distortion of DNA (helices) at fork
What are the non-essential elements for the rep O?
BS for activators/ chromatin remodellers
What is the role of the origin Rep Complex? and what does it form?
recruit other replication p to the origin & helicase
form pre-replactitive complex
what assists helicases in opening the strand?
- Start site binding P
- Cdc45 and GINS bind to MCM complex and make it active.
- only occurs when S phase goes ahead.
- Protect & stabilise exposed start site DNA templates.
What are the first things that bind to the origin of rep in EUK and PRO?
PRO -> initiator P
EUK -> origin recognition complex ORC
Recruit the DNA helicases to the origin region.
What binds to the origin of rep first?
ORC
origin rep complex
What proteins make up the helicases molecules?
active vs inactive
inactive =MCM hexamer- Cdt1 complex
Active= CMG helices complex, Cdc 45, MCM + GINS
2 bind and when S phase is initiated they separate to the each strand
How many proteins make up ORC?
6
What is the shape of the helicase?
ring-shape and hexameric
similar in PRO and EUK
What is the section that separates the DNA strands?
NTP hydrolysis
How does helicase move and unwrap DNA?
reaction?
NTP hydrolysis
change shape of protein, move rapidly along DNA strand and unwrap as it goes.
What McM subunits bind and create a gate between with the ORC-Cdc6 complex?
2-5
What element does DNAp need to bind to DNA?
priming group
What is the function of the priming group?
provides the 3’OH group paired to DNA template required by DNA polymerases to initiate Replication
How can priming be achieved?
- nick the DNA at origin to pose the 3’OH group
- pre-formed RNA acts as a primer
- primase enzymes synthesise RNA primers in PRO and EU
What enzyme synthesis RNA primers?
primases
What are primases?
small RNA polymerases
binds to NTP
Creates a short stretch or RNA with functions as the primer (chain of ribonuclease)
Is the RNA polymerase removed?
YES, it is replaces with DMAp with high fidelity
transiently associated with DNA
PRO primase?
DnaG
has a Zn2+ binding region for protein-protein/ protein-DNA binding
Where does the palm on the DNAp bind to on the DNA?
palm binds the primer template junction and the newly synthesised DNA
through interactions with the phosphate backbone of the minor groove.
What mediates DNTP binding?
by base pairing through H bonding and binding to a divalent metal cation (Mg2+)
What is the role of the finger region of DNAp?
closes around the bound dNTP, prevents hydrolysis and allows nuceophillic attack of oxygen of the 3’ hydroxyl on the alpha phosphate to form a phospodiesteir bond.
What can cause spontaneous mutation?
misfiring to tautness during DNA rep
occur very infrequently
Do DNAp have proofreading activity?
YES!
What is exonuclease?
enzymes that work by cleaving nucleotides one at a time from the end of the polynucleotide chain
What reaction does a exonuclease undergo?
hydrolysis
breaks the phosphate bonds
What are the DNAp in E.coli? PRO
DNA i - doesn’t replicate most of the DNA
DNAp(iii)
how many pol3 are involved in e.coli rep?
3
1 for leading and 2 for Okazaki segments
How are the RNA primers removed in PRO?
POL1
last step
exonucleases
and uses RNAp to synthesise gaps
What DNAp are involved in DNA synthesis?
alpha
delta
epsilon
What sub of the alpha DNAp synthesises the initiator complex? euk
alpha sub
3’ OH of initiator DNA is used to prime DNA synthesis of the DNAp epsilon and delta.
What POL synthesis leading nd lagging stands?
leading epsilon
lagging delta
3’->5’ exonuclease proof reading activity
What elements are RNA primers removed at the end of DNA rep? EUK
combined activity of DNAp and Fen1 (flap endonuclease 1)
How does DNAp displace RNA primers? EUK
It extends the fragments until it displaces the RNA primers, forming a flap
Fen1 removes this flap (its an endonuclease)
How does DNA ligase work? (general)
catalyses formation of a phosphodiester bond between the adjacent 3’OH and 5’P ends of the polynucleotides
H-bonded to a complementary strand
How does DNA ligase work in EUK?
enzyme nucleotide intermediate
What is adenylyl
.
How to topoisomerases reduce positive supercoils?
formation of covalent protein-DNA intermediate allowing the DNA to rotate and re-ligate one or both strands to prevent torsional strain
POL can only hold on to the DNA for a small amount of time (low processivity), what factors aid the prolonged binding? PRO/EUK
A clamp is required to increase the processivity of P
can synthesise long DNA strands
What is the processivity of POLiii like? PRO
low, catalyses ~11phosdiester bonds
unbinds and binds repeatedly
What enzyme is associated with POLiii to increase processivity?
HOLO enzyme
addition of many subunits
What makes up the HOLO enzyme processive? PRO/EUK
many subunits but the last to attach is the clamp and makes the enzyme highly processive
catalyses thousands of phosopdiester bonds before dissociating
What subunit is Clamp on HOLO enzyme? PRO
beta
What is more processive POL1 OR POL3? E.coli PRO
3 as it has holo enzyme so does not dissociate from DNA as frequently
no loss of accuracy
What is the processivity factor in EUK?
PCNA
How do clamps associate with DNA?
the aid of clamp loader
What are clamp loaders?
ATPases
What are telomeres?
tandem repeats of a short conserved seq with a block of G nucleotides
What is telomerase ?
essentially a reverse transcriptase
a DNA polymerase, synthesises DNA using an RNA template
When are tolomerases present in the cell cycle?
on the ends of chromosome at the end of G1 phase, active during S phase
